Malaria caused by Plasmodium falciparum results in over a million deaths a year in Africa alone. Drug resistant strains of the parasite are common. A chemotherapeutic target of current interest is the mitochondrion. The goal of the proposed study is to test the hypothesis that the mitochondria of P. falciparum utilize a cyanide-resistant alternative respiration pathway as well as the classical, cyanide-sensitive, respiration pathway. Utilization of the alternative respiratory pathway in P. falciparum is indicated by preliminary evidence that parasite oxygen consumption and mitochondrial transmembrane potential can be rapidly and completely eliminated only by a combination of inhibitors that act on both the classical and alternative respiratory pathways. Immunological evidence suggesting the developmental regulation of the key protein required for alternative respiration, the alternative oxidase, is supported by inhibitor studies. The proposed project has three specific aims. The first is to determine the physiological properties of the alternative respiratory pathway in P. falciparum. In particular, potential chemical and environmental inducers of the alternative respiratory pathway will be tested for their ability to increase cyanide-resistant oxygen consumption. In the second specific aim, the potential utility of targeting the alternative respiratory pathway for chemotherapeutic intervention will be assessed. Preliminary evidence for parasite growth inhibition by inhibitors of the alternative oxidases of other organisms will be expanded by testing parasites from different geographic origins and drug resistance backgrounds. The mechanism of action of these inhibitors in P. falciparum will also be tested. In addition, the hypothesis that a mixture of inhibitors of the alternative and classical respiratory pathways will act synergistically in the parasite will be tested. The third specific aim is to clone the gene for P. falciparum alternative oxidase. The cloned gene will be used to measure developmental regulation of the parasite alternative oxidase. Finally, expression of the P. falciparum alternative oxidase gene in E. coli will be used to test for its activity in this heterologous system and as a first step toward the generation of immune sera to parasite alternative oxidase.